安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
Acta Universitatis Medicinalis Anhui
2015年
11期
1560-1565
,共6页
何汉文%王葱%杜博易%吴璇%于莉%汪%黄升海
何漢文%王蔥%杜博易%吳璇%于莉%汪%黃升海
하한문%왕총%두박역%오선%우리%왕%황승해
呼吸道合胞病毒%A549细胞%Toll样受体7%siRNA%I型干扰素
呼吸道閤胞病毒%A549細胞%Toll樣受體7%siRNA%I型榦擾素
호흡도합포병독%A549세포%Toll양수체7%siRNA%I형간우소
respiratory syncytial virus,A549 cells%Toll-like receptors 7%siRNA%Type I interferon
目的:探讨 Toll 样受体7(TLR7)在呼吸道合胞病毒(RSV)感染 A549细胞诱导产生 I 型干扰素(IFN)抗病毒免疫反应中的作用。方法实验分正常对照组、RSV 感染组、TLR7 siRNA 沉默组,分别于感染的4、8、12、24 h 后收集各组细胞以及培养上清液。TLR7 siRNA 通过瞬时转染 A549细胞,半定量 RT-PCR 法检测 TLR7基因沉默效果;TRIzol 试剂提取细胞总 RNA,检测 TLR7、干扰素调节因子7(IRF7)、IFN-α、IFN-β的 mRNA 表达量变化;通过 Western blot 法检测 TLR7蛋白表达量;ELISA 法检测感染不同时间点 A549细胞培养上清液中 IFNα/β含量的变化。结果①转染24 h 后 TLR7 siRNA-2有效抑制 TLR7 mRNA 表达;② RSV 感染A549细胞后,TLR7、IRF7、IFN-α/β的 mRNA 以及 TLR7蛋白的表达量均升高,并与 RSV 感染之间存在时间依赖性关系;沉默 TLR7后,TLR7、IRF7、IFN-α/β的 mRNA 以及 TLR7蛋白的表达量较感染组均有明显下降;③ RSV 感染 A549细胞后,明显上调细胞培养上清液中 IFN-α/β的水平,TLR7 siRNA 沉默组 IFN-α/β的表达水平较 RSV 感染组均下降,差异有统计学意义,且 IFN-α的表达量下调更为显著。结论RSV 感染A549细胞后,TLR7被活化能够诱导产生I 型IFN,发挥抗病毒作用,且TLR7诱导产生的I 型IFN 以IFN-α为主。
目的:探討 Toll 樣受體7(TLR7)在呼吸道閤胞病毒(RSV)感染 A549細胞誘導產生 I 型榦擾素(IFN)抗病毒免疫反應中的作用。方法實驗分正常對照組、RSV 感染組、TLR7 siRNA 沉默組,分彆于感染的4、8、12、24 h 後收集各組細胞以及培養上清液。TLR7 siRNA 通過瞬時轉染 A549細胞,半定量 RT-PCR 法檢測 TLR7基因沉默效果;TRIzol 試劑提取細胞總 RNA,檢測 TLR7、榦擾素調節因子7(IRF7)、IFN-α、IFN-β的 mRNA 錶達量變化;通過 Western blot 法檢測 TLR7蛋白錶達量;ELISA 法檢測感染不同時間點 A549細胞培養上清液中 IFNα/β含量的變化。結果①轉染24 h 後 TLR7 siRNA-2有效抑製 TLR7 mRNA 錶達;② RSV 感染A549細胞後,TLR7、IRF7、IFN-α/β的 mRNA 以及 TLR7蛋白的錶達量均升高,併與 RSV 感染之間存在時間依賴性關繫;沉默 TLR7後,TLR7、IRF7、IFN-α/β的 mRNA 以及 TLR7蛋白的錶達量較感染組均有明顯下降;③ RSV 感染 A549細胞後,明顯上調細胞培養上清液中 IFN-α/β的水平,TLR7 siRNA 沉默組 IFN-α/β的錶達水平較 RSV 感染組均下降,差異有統計學意義,且 IFN-α的錶達量下調更為顯著。結論RSV 感染A549細胞後,TLR7被活化能夠誘導產生I 型IFN,髮揮抗病毒作用,且TLR7誘導產生的I 型IFN 以IFN-α為主。
목적:탐토 Toll 양수체7(TLR7)재호흡도합포병독(RSV)감염 A549세포유도산생 I 형간우소(IFN)항병독면역반응중적작용。방법실험분정상대조조、RSV 감염조、TLR7 siRNA 침묵조,분별우감염적4、8、12、24 h 후수집각조세포이급배양상청액。TLR7 siRNA 통과순시전염 A549세포,반정량 RT-PCR 법검측 TLR7기인침묵효과;TRIzol 시제제취세포총 RNA,검측 TLR7、간우소조절인자7(IRF7)、IFN-α、IFN-β적 mRNA 표체량변화;통과 Western blot 법검측 TLR7단백표체량;ELISA 법검측감염불동시간점 A549세포배양상청액중 IFNα/β함량적변화。결과①전염24 h 후 TLR7 siRNA-2유효억제 TLR7 mRNA 표체;② RSV 감염A549세포후,TLR7、IRF7、IFN-α/β적 mRNA 이급 TLR7단백적표체량균승고,병여 RSV 감염지간존재시간의뢰성관계;침묵 TLR7후,TLR7、IRF7、IFN-α/β적 mRNA 이급 TLR7단백적표체량교감염조균유명현하강;③ RSV 감염 A549세포후,명현상조세포배양상청액중 IFN-α/β적수평,TLR7 siRNA 침묵조 IFN-α/β적표체수평교 RSV 감염조균하강,차이유통계학의의,차 IFN-α적표체량하조경위현저。결론RSV 감염A549세포후,TLR7피활화능구유도산생I 형IFN,발휘항병독작용,차TLR7유도산생적I 형IFN 이IFN-α위주。
Objective This experiment was designed to explore the antiviral effect of TLR7 when A549 cells were infected by respiratory syncytial virus (RSV)to induce the production of type I interferon (IFN).Methods This experiment was divided into normal control group,RSV infection group and TLR7 silence group.In each group cell and culture supernatant were collected after having been infected for 4,8,12 and 24 h.TLR7 siRNA was trans-fected in A549 cells by transient transfection.With the use of the RT-PCR assay,TLR7 gene silencing effect could be detected;with the use of Trizol reagent,the total RNA could be extracted and the expression dynamics of mRNA in TLR7,IRF7,IFN-αand IFN-βcould be detected;with the use of Western blot,protein expression could be tested;with the use of ELISA,the variation of IFNα/βin the culture supernatant of A549 cells infected in the dif-ferent points of time could be detected.Results ① 24 h after transfection,TLR7 siRNA-2 could restrain the ex-pression of TLR7 mRNA effectively with statistical significance.② The expression of mRNA in TLR7,IRF7 and IFN-α/β,together with TLR7 protein,which existed time dependent relation with RSV infection,had increased af-ter A549 cells had been infected by RSV.Meanwhile,compared with RSV infection group,the expression of mR-NA in TLR7,IRF7 and IFN-α/β,together with TLR7 protein had decreased obviously in TLR7 silence group.③Compared with RSV infection group,the expression of IFN-α/βin cell culture supernatant and in TLR7 silent group had decreased,and the expression of IFN-αdecreased obviously.It went without saying that the difference of decrease in two groups had statistical significance.Conclusion After cell A549 have been infected by RSV, TLR7 could be activated and then induce type I interferon after having been infected by RSV,which play the role in anti-virus.Noticeably,IFN-αaccounts for the most of e type I interferon.